Vitamin D analogues

ABSTRACT

The present invention relates to compounds of formula (I), in which formula X is hydrogen or hydroxy; R 1  and R 2 , which may be the same or different, stand for hydrogen or a C 1 -C 3  alkyl radical; or R 1  and R 2 , taken together with the carbon atom bearing the group X, can form a C 3 -C 6  carbocyclic ring, R 3  stands for a C 1 -C 3  alkyl radical, an aryl or an aralkyl radical, or for YR 4 , in which Y stands for the radicals —CO—S—, —CS—O— or —CS—S—, and R 4  stands for a C 1 -C 3  alkyl radical or an aryl or an aralkyl radical; Q is (CH 2 ) n , n being 1-4. R 1 , R 2  and Q independently may optionally be substituted with one or more fluorine atoms. The compounds show strong activity in inducing differentiation and inhibiting undesirable proliferation of certain cells as well as immunomodulating and antiinflammatory effects.

This application is the national phase of international applicationPCT/DK97/00225 filed May 5, 1997.

This invention relates to a hitherto unknown class of compounds whichshows strong activity in inducing differentiation and inhibitingundesirable proliferation of certain cells, including skin cells andcancer cells, as well as immunomodulating and antiinflammatory effects,to pharmaceutical preparations containing these compounds, to dosageunits of such preparations, and to their use in the treatment and/orprophylaxis of diseases characterized by abnormal cell differentiationand/or cell proliferation such as e.g. psoriasis and other disturbancesof keratinization, HIV-associated dermatoses, wound healing, cancer,including skin cancer, and of diseases of, or imbalance in, the immunesystem, such as host versus graft and graft versus host reaction andtransplant rejection, and autoimmune diseases, such as discoid andsystemic lupus erythematosus, diabetes mellitus and chronic dermatosesof autoimmune type, e.g. scleroderma and pemphigus vulgaris, andinflammatory diseases, such as rheumatoid arthritis and asthma, as wellas a number of other disease states including hyperparathyroidism,particularly secondary hyperparathyroidism associated with renalfailure, cognitive impairment or senile dementia (Alzheimers disease)and other neurodegenerative diseases, hypertension, acne, alopecia, skinatrophy, e.g. steroid induced skin atrophy, skin ageing, includingphoto-ageing, and to their use for promoting osteogenesis andtreating/preventing osteoporosis and osteomalacia.

The compounds of the invention constitute a novel class of vitamin Danalogues and are represented by the general formula I

in which formula X is hydrogen or hydroxy; R¹ and R², which may be thesame or different, stand for hydrogen or a C₁-C₃ alkyl radical; or R¹and R², taken together with the carbon atom bearing the group X, canform a C₃-C₆ carbocyclic ring; R³ stands for a C₁-C₃ alkyl radical, anaryl or an aralkyl radical, or for YR⁴, in which Y stands for theradicals —CO—S—, —CS—O— or —CS—S—, and R⁴ stands for a C₁-C₃ alkylradical or an aryl or an aralkyl radical; Q is (CH₂)_(n), n being 1-4.R¹, R² and Q independently may optionally be substituted with one ormore fluorine atoms.

Examples of R¹ and R² when taken separately include (apart fromhydrogen), but are not limited to, methyl, trifluoromethyl, ethyl,pentafluoroethyl, and normal-, iso- and cyclo-propyl.

Examples of R¹ and R² when taken together include di-, tri-, tetra- andpentamethylene.

Examples of R³ and R⁴ include, but are not limited to, methyl, ethyl,propyl, normal-, iso- and cyclopropyl, phenyl and benzyl.

Most preferred examples of Q include di- and trimethylene.

As can be seen from formula I, depending on the meanings of R¹, R², R³,Q and X the compounds of the invention can comprise severaldiastereoisomeric forms (e.g. R or S configuration at the carbon atombearing the radicals R¹, R², and X). The invention covers all thesediastereoisomers in pure form as well as mixtures thereof.

In particular, both diastereoisomers having the two possibleconfigurations (in the following designated “A” and “B”) at the carbonatom marked “22” are included. A is the preferred one.

In addition, prodrugs of I in which one or more of the hydroxy groupsare masked as groups which can be reconverted to hydroxy groups in vivocan also be envisaged.

Compounds of formula I in which X is hydrogen also may act as prodrugs,as these compounds are relatively inactive in vitro but are converted toactive compounds of formula I by enzymatic hydroxylation afteradministration to the patient.

It has been known for more than two decades that vitamin D (D₃/D₂) is apro-hormone which is hydroxylated in vivo to the active hormone1α,25-dihydroxy-vitamin D₃ (1,25(OH)₂D₃ or calcitriol). Vitamin D₂ ismetabolized in the same way as D₃ and is bioequivalent with D₃ inhumans. 1,25(OH)₂D₃ is responsible for maintaining calcium (andphosphate) homeostasis by regulation of the intestinal calciumabsorption, the renal calcium excretion and the bonemineralization/resorption. Other hormones such as parathyroid hormone(PTH) and calcitonin function as positive and negative feedback calciumregulators respectively in concert with 1,25(OH)₂D₃ [1],[2].

This knowledge has led to the use of 1,25(OH)₂D₃ and its prodrug1α-hydroxy-D₃ (alfacalcidol, INN) in the treatment of patients withkidney failure, where the important renal 1α-hydroxylation of 25-hydroxyvitamin D is impaired, leading to hypocalcemia, secondaryhyperparathyroidism and bone demineralization (renal osteodystrophy)[1].

It has since been shown that the (nuclear) receptor for 1,25(OH)₂D₃(VDR) is present, not only in the intestine, bone and kidney, but in alarge number of other locations, such as e.g. parathyroid glands, isletsof the pancreas, mammary gland cells, keratinocytes and fibroblasts ofthe skin, circulating monocytes and (activated) lymphocytes, and manyother normal cell types and tissue; in addition the VDR is also presentin several cancer cell lines [2],[3],[4].

In accordance with this widespread occurrence of the VDR, it has beenexperimentally demonstrated that 1,25(OH)₂D₃ has biological effects overand beyond the “classical” effects on calcium regulation.

It was shown that 1,25(OH)₂D₃ is able to stimulate the differentiationof cells and inhibit excessive cell proliferation [5], suggestingpotential use in the treatment of e.g. psoriasis and cancer. It was alsoshown that 1,25(OH)₂D₃ influences the effects and/or production ofinterleukins [6], indicating the potential use of this compound in thetreatment of immunological disorders, such as e.g. autoimmune diseasesand rejection of transplants.

The use of 1,25(OH)₂D₃, or its pro-drug 1α(—OH—D₃, for the treatment ofseveral other disease states has also been suggested: hypertension [7],diabetes mellitus [8], alopecia [9], acne [10], osteoporosis [11] andneurodegenerative disorders [121.

The inhibition of angiogenesis by 1,25(OH)₂D₃[13] indicates a possibleinhibition of tumour growth via reduction of the growth of new bloodvessels into the tumour.

However, the therapeutic possibilities of 1,25(OH)₂D₃ in several ofthese indications are severely limited by the potent effect of thishormone on calcium metabolism, because serious side effects due tohypercalcemia will result from the high doses necessary to obtain atherapeutic effect on e.g. psoriasis or cancer or immunologicaldisorders.

In order to overcome this problem a large number of vitamin D analogueshave been described, and some of these show selectivity in favour of thecell differentiation inducing/cell proliferation inhibiting activity ascompared with the effect on calcium metabolism [3],[4],[14].

This work has resulted in therapeutically useful, or potentially useful,vitamin D analogues. Thus, the vitamin D₃ analogue, MC₉₀₃ (calcipotriol,calcipotriene, INN, cf. table 1), is a potent inducer of celldifferentiation and inhibitor of cell proliferation which shows onlymoderate activity on calcium metabolism in vivo [15].

Calcipotriol is on the market as a safe and effective drug for thetreatment of the hyperproliferative disease of the skin, psoriasis.Calcipotriol renders improvement, without hypercalcemia, in 70-80% ofthe patients ([6],[17]. However, this selectivity is not paralleled byin vitro studies, which show that calcipotriol binds equally well as1,25(OH)₂D₃ to the intestinal vitamin D receptor. The low in vivoactivity on calcium metabolism of calcipotriol is due to a rapidmetabolism of the compound, thus limiting the potential of this compoundfor systemic use [16].

The efficacy of several vitamin D analogues in inducing differentiationand inhibiting proliferation of cancer cells has been demonstrated, bothin vitro and in vivo see e.g. [3], [4], [11], [14], [18], [19], [20],[21], [22], [23].

One such interesting analogue is EB 1089 [22], [24] which inhibits thegrowth of breast cancer cells in vitro and in vivo [4], [25]. There is adefinite selectivity between the anti-cancer and the calcemic effects invivo, but the molecular basis for this selectivity is not reallyunderstood [4]. EB 1089 is presently in clinical study [26].

A promising series of vitamin D analogues having differentiationinducing and antiproliferative activity are the 20-epi-analogues [18],[27], [28] in which the stereo-chemistry at carbon-20 in the side chainis altered to the “unnatural”, so-called 20-epi, configuration. The twomost studied of these 20-epi-analogues are KH 1060(20-epi-22-oxa-25,26,27-trihomo-1,25-(OH)₂D₃; lexacalcitol, INN) [18],[29] and MC₁₂₈₈ (20-epi-1,25(OH)₂D₃; cf. table 1) [18], [30].

It was found that the change from 20-“normal” to 20-“epi” configurationhas a profound and advantageous biological significance. Generallyspeaking, the most active of these analogues are characterized by theirhigh, or very high, potencies as regulators of cell growth and/or immuneresponses; their calcemic activity being equivalent to that of1,25(OH)₂D₃, or a few times stronger [18].

Separation of immunoregulating activity from calcemic activity has alsobeen demonstrated for some vitamin D analogues [18], [27]. Someimportant findings concerning the immunological effects of 1,25(OH)₂D₃itself will be illustrative of the present state of the art; referencesto original papers can be found in recent articles, e.g. [27], [31] and[32] (dermatology in particular). 1,25(OH)₂D₃ stimulates thedifferentiation of monocytes towards macrophages and enhances theirantibacterial activity, and 1,25(OH)₂D₃ restores normal immune functionsin vitamin D deficiency states in vivo. On the other hand 1,25(OH)₂D₃has an immunosuppressive effect on the antigen-mediated part of theimmune system. The proliferation of activated T-lymphocytes is inhibitedwith down-regulation of cytokines IL-2 and INF-γ. In vitro thisdown-regulation leads to an indirect inhibition of the formation ofantibodies (IgM and IgG) from B-lymphocytes. Cytotoxic T-cells are alsoinhibited and regulatory/suppressor T-lymphocytes are stimulated. Somestudies also report downregulation by 1,25(OH)₂D₃ of theproduction/release of cytokines such as IL-1 and TNF-C fromantigen-stimulated monocytes/macrophages.

These findings suggest clinical applications of 1,25(OH)₂D₃ in thefields of organ or cell transplantations and in autoimmune diseases, butits calcemic effects severely limit its usefulness in clinical practice.

The new vitamin D analogues [18], [27] however, show some promise inthis direction, as the following examples will indicate.

The 20-epi-analogue MC₁₂₈₈ was effective as an immunosuppressant in arat model of heart and small bowel transplantations, preventing graftrejection [30]; the serum calcium level was moderately increased.

The 20-epi- and the 20-normal analogues KH 1060 and CB 966 prolongedskin allograft survival in mice [33]. KH 1060 was the most potentimmunosuppressor, but induced hypercalcemia in the highest, and mosteffective, dose. Combination treatment with Cyclosporin A (CyA) was moreeffective than either agent alone, with moderate rise of serum calcium.MC 1288 and two other 20-epi-analogues were also very effective in thisassay [34e] and were less hypercalcemic than KH 1060.

KH 1060 was an effective immunoregulator in the autoimmune disease, typeI diabetes, which was studied in the non-obese diabetic (NOD) mouse[35]. The effect on calcium and bone was only slight, in contrast to1,25(OH)₂D₃ (which otherwise was an effective immunomodulator, too).

In a related study [36] where the effect of autoimmune memory wasstudied by the transplantation of completely MHC¹-matched, or syngeneic,—cells of the islets of the pancreas to NOD mice, both KH 1060 and CyAwere very effective in delaying autoimmune disease recurrence, but bothwere toxic in the most effective high doses. KH 1060 and CyA combined,in lower, nontoxic, doses were as effective as each compound in highdose, and the effect on calcium metabolism was reduced.

¹major histocompatibility complex

There is recent evidence [37] which indicates that the powerful activityof the 20-epi-analogues KH 1060 and MC₁₂₈₈ may be connected withenhanced transcriptional activity, compared to 1,25(OH)₂D₃, but in vivothere is a long range of other factors, such as e.g. absorption,transport to target cells (binding proteins) and metabolic and cellularuptake properties, that may determine the non-classic versus calcitropicactivities of vitamin D analogues [11], [14](p.301), [20], so that theselection of potential candidates for clinical application cannot solelybe based on in vitro screening.

As will be seen from the above description of the present state of theart, the newer vitamin D analogues are quite effective agents fortreating psoriasis and they are promising as immunosuppressive andanticancer agents. However, there is still a need for an improvedefficacy and a higher safety ratio in respect to the calcitropic sideeffects.

The compounds of the present invention have new and unexpectedadvantageous properties, which mean that they are promising agents,possessing increased efficacy or reduced side effects, for treating suchpathological states for which 1,25(OH)₂D₃ or its analogues would beindicated, but for their calcemic (side) effect.

The topic of the relationship between the chemical structure of vitaminD analogues and their biological activity (in vitro or in vivo) has beendiscussed; for recent reviews see e.g. [3], [21]. For newer analoguesfrom LEO, see [34a-d] and [38a-d]; in these studies systematicstructural modifications has been correlated with inhibition ofproliferation and induction of differentiation of U 937 leukaemia cells,with calcemic activity in the rat, and with vitamin D receptor binding.The structural modifications include: side chain length and branching[34a-d], [38a-d], do.-unsaturation [34a,b,c], (38c,d],C-20-stereochemistry [34a,b,c], [38b,c], heteroatoms or aromatic ringsin the side chain [34b], [38b] and hydroxy or alkoxy substitution atC-20 [34d], C-22 [34c] and C-24 [38c]. It appears that there isfrequently very big variation in the activity, even for small structuralchanges [34a,d], [38b,c]. In most cases there is little relationshipbetween in vitro anti-cancer activity and in vivo calcemic activity, orreceptor binding affinity. Some receptor binding is evidently necessaryfor cell regulating activity [27], but e.g. in the case of 20- and22-hydroxy/alkoxy substituted analogues, potent anti-cancer activity iscompatible with very low receptor binding [34c,d]. Correlations betweenstructure and immunological activity has also been discussed [18], [27];it is found that some of the 20-epi analogues are potentimmunosuppressive agents.

Summarizing this evidence: while it may be possible, within a confinedseries of very closely related compounds, to some extent to correlate astructural change with the result from a well-known biological assay, itis impossible to make predictions of the activity in a new assay fromthe chemical structure alone.

Surprisingly, compounds 101 and 102 of the present invention have newand unexpected biological activity in the assays presented in table 1,in which table also the corresponding activities of some closely relatedreference compounds are included. The calcemic activities of thereference compounds have been described [15], [18], [34c], and alsoMLR-data for MC 1288 [18], but it is apparent that the potent effects ofthe compounds of the invention on skin cell proliferation and mixedlymphocyte reaction combined with only moderate calcemic activity couldnot be predicted from these prior art data.

European patent application 0 296 800, filing date Jun. 21, 1988,discloses compounds which are vitamin D analogues containing asaturated, all carbon, side chain, substituted at position 22 with ahydroxy or a lower alkyloxy group and these compounds are claimed to beuseful in the treatment of disease states characterized by metaboliccalcium deficiencies or having tumour cell differentiation-inducingactivity. Similar compounds are also described in the non-patentliterature [39], [40]. They were tested for vitamin D activity, whichthey did not have, i.e. no significant hypercalcemic effect in rats[39], and for their ability to induce differentiation of HL-60 cancercells. The most active compound was 22S-methoxy-1,25(OH)₂D₃ which was0.25 times as potent as 1,25(OH)₂D₃ in this assay. The receptor bindingaffinity of 22S-methoxy-1,25(OH)₂D₃ was 0.03 times that of 1,25(OH)₂D₃,[40].

The compounds I of the present invention differ from these prior artcompounds by having the above mentioned “unnatural”20-epi-configuration. This configuration is also present in severalother newer vitamin D analogues, among them the compounds disclosed inour previous international patent application number PCT/DK90/00156,filing date Jun. 19, 1990, publication number WO 91/00271, andinternational patent application number PCT/DK93/00105, filing date Mar.23, 1993, publication number WO 93/19044, and in Japanese patentapplication, publication number 7-304733, filing date May 10, 1994.These 20-epi-analogues are stated to have cell differentiation inducingactivity etc.

The present compounds I are distinguished from the compounds ofPCT/DK90/00156 and Japanese patent application 7-304733 in having analkyloxy or alkyl(aryl)oxy carbonyloxy (or thio-/dithio-alkyl(aryl)oxycarbonyloxy) substituent in position 22 of the side chain, instead ofhydrogen atoms or a hydroxy group or a protected hydroxy group in thisposition, and are distinguished from the compounds of PCT/DK93/00105which carry a carbon-carbon triple bond in position 23,24 of the sidechain, in having only carbon-carbon single bonds in the side chain. Thestructural differences of the present compounds I from the related20-epi vitamin D analogues of the above mentioned patent applicationsmade it possible to achieve new and unexpected advantages, asdemonstrated in the biological assays mentioned in the following.

In order to demonstrate the effectiveness of the compounds of formula Iof the invention, the information of Table 1 is referred to: inparticular columns “HaCaT, rel.”, “HaCaT, max. %”, “MLR, rel.”, and“Calc., rel.”; the meaning of which is explained in the following.

A useful assay for the rating of test compounds for antiproliferativeactivity in skin cells, e. g. antipsoriatic effect, is the in vitroassay using HaCaT, a spontaneously immortalized, non-tumorigenic humanskin keratinocyte cell line, [41], measuring ³H-thymidine uptake.

An in vitro assay for the rating of test compounds for immunosuppressivepotency is the mixed lymphocyte reaction assay, “MLR”, measuring theallogeneic stimulation of mouse spleen lymphocytes: lymphocytes,obtained from the spleens of BALB/c and CB6F1 mice, are stimulated byco-cultivating 5×10⁶/ml cells from BALB/c mice (responders) with7.5×10⁶/ml cells from CB6F1 mice (inducers). The mixed cultures oflymphocytes are incubated with the test compounds for 72 hours. CellularDNA-synthesis is assessed by the incorporation of ³H-thymidine in the D

Generally, the classical effects of 1,25(OH)₂ vitamin D₃ on the calciumbalance in the organism, including calcemic and calciuric activities,are unwanted in the vitamin D analogues of the present invention, inwhich selectivity for e.g. inhibition of the proliferation of certaincells and/or immunosuppressive activity is normally desired.

The calcemic activity of the compounds was determined in rats in vivo,as previously described [15]. In table 1, column “Calc., rel.”, thecalcemic activities of selected compounds (relative to 1,25(OH)₂D₃) arelisted; as mentioned, low values for the compounds of the presentinvention are ordinarily preferred.

It appears from table 1 that the two selected exemplified compounds,Comp. 101 and Comp. 102 are considerably more potent than 1,25(OH)₂D₃ inthe HaCaT-assay (psoriasis model), while retaining the same, or higher,maximal inhibition at 10⁻⁷ M as 1,25(OH)₂D₃. In addition the calcemicactivity is only about half as large as that of 1,25(OH)₂D₃.

Concerning the other important property of the compounds I of theinvention, their immunosuppressive activity, it is apparent from Table1, column “MLR, rel.”, that the selected Compounds 101 and 102 havepotent effects, also in comparison with the prior art referencecompounds. Of these, the compound MC1288 is particularly interesting, asit has been demonstrated to have a valuable immunosuppressive activityin animal transplantation experiments [30]. In addition the compounds ofthe present invention exhibit a favourable ratio between the relativeMLR-potency and the relative calcemic activity.

Thus, also when the immunosuppressive properties are considered, lessdanger of calcemic side effects is to be expected from the presentcompounds 1, than from those of prior art mentioned in table 1.

TABLE 1 Biological Tests of Compounds I and Reference Compounds HaCaTHaCaT MLR Calc. Side chain at C-17 Comp. Code rel. max. % rel. rel. οNo. No. ¤, ¶ • ¤, ¶ ¤

101 28 92 175 0.45

102 137 85 161 0.50 Reference compounds

1,25* 1 85 1 1

MC903# 1.8 78 0.6 0.005

MC1288 43 76 150 2.5

CB1297 45 75 12 n.d.

GS1535 313 82 13 8.2

GS1725 4.4 96 150 12

GS1720 3.9 70 36 1.2 Notes to Table 1 ο The rest of the molecule is thesame as in formula I. The 22-OH/OMe/OEt compounds are all 22A-isomers. ¤The values are relative to 1,25 (OH)₂D₃; a value greater than 1indicates a compound which is more active than 1,25 (OH)₂D₃ in theassay. ¶ Calculated as the ratio between the IC₅₀ value of 1,25 (OH)₂D₃and the IC₅₀ value of the compound; IC₅₀ being the concentration whichresults in 50% inhibition of the ³H-thymidine incorporation compared tocontrols. • “HaCaT, max %” indicates the maximal inhibition of the cellproliferation (maximal efficacy) which is obtainable at the higherconcentration of 10⁻⁷ M of a compound. * 1,25 = 1,25 (OH)₂D₃ = 1,25(OH)₂-vitamin-D₃. # MC903 = Calcipotriol, (see text above). n.d. = notdetermined.

Compounds I can be prepared from the vitamin D-derived aldehyde compound1 (Scheme 1); a synthesis of which has been reported [42], for exampleby the routes outlined in Scheme 1.

The following standard abbreviations are used throughout thisdisclosure: Me=methyl; Et=ethyl; Pr=n-propyl; Bn=benzyl; Ph=phenyl;THP=tetrahydro-4H-pyran-2-yl; TMS=trimethylsilyl;DMAP=4-dimethylaminopyridine; PPTS=pyridinium p-toluenesulfonate;pet.ether=petroleum ether; THF=tetrahydrofuran;TBAF=tetra-(n-butyl)-ammonium fluoride; b.p.=boiling point;PLC=Preparative thin-Layer Chromatography; HPLC=High Performance LiquidChromatography.

X¹=H, OH, OR⁵

R⁵=alcohol protective group, e.g. tri(loweralkyl)-silyl or THP

R¹, R², R³, Q and X have the above meanings.

Notes to Scheme 1

a) (i) Compound 1 is reacted with an organometallic reagent R-Met-Hal orR-Met, such as e.g. RMgHal, where Hal is Cl, Br or I, or RLi, which maymay be prepared from the side chain building block of general formula V,RHal, (see below) by reaction with a suitable metal, such as magnesiumor lithium.

(ii) The resulting mixture of the two C-22-epimers, IIA and IIB, isseparated.

b) Alkylation of the C-22-hydroxy compounds of type 11 to thecorresponding compound III, where R³=C₁-C₁₀ hydrocarbyl, or, optionally,acylation of the compounds of type II to the corresponding compound III,where R³=YR⁴; Y and R⁴ having the above meanings.

c) Isomerization of Compounds III to the corresponding compound IV, bymeans of UV-light in the presence of a triplet sensitizer, e.g.anthracene.

d) Deprotection of Compounds IV to the corresponding compound I, e.g. byTBAF, by HF, or by TBAF followed by PPTS, or vice versa.

The compounds of type II or III may be subjected to optional functionalgroup modification in the side chain, if considered desirable ornecessary during a particular synthesis.

The order of the synthetic steps, which in Scheme 1 is: a), b), c), d),may, if for any reason desirable, be rearranged to the order: a), c),b), d), or the order: a), b), d), c).

The side chain building blocks of general formula V, RHal, are knowncompounds, or they can be prepared by standard methods known to thespecialist.

HaI—Q—CR¹R²—X¹

in which HaI, Q, R¹, R² and X¹ have the above meanings. In particular,this applies to the side chain building blocks of general formula Vnecessary for the preparation of the exemplified compounds (101-108,110-112, 114-116 and 121); these RHaI are described in table 2. Othersimilar RHaI may be prepared by methods analogous to those used forsynthesizing the compounds V of table 2. For X¹=O—THP, see e.g. WO93/19044.

TABLE 2 Some Side Chain Building Blocks, R-Hal of General Formula VCompound Formula References 501 Br(CH₂)₂C(CH₃)₂OSi(CH₃)₃ WO 91/00271 502Br(CH₂)₂C(C₂H₅)₂OSi(CH₃)₃ WO 94/14766 503 Br(CH₂)₃C(CH₃)₃OSi(CH₃)₃ WO91/15475 504 Br(CH₂)₃C(C₂H₅)₂OSi(CH₃)₃ WO 89/10351 505Br(CH₂)₄C(C₂H₅)₂OSi(CH₃)₃ WO 89/10351

The reaction of the aldehyde 1 with the organometallic reagents derivedfrom the side chain building blocks, e.g. RMgHaI or RLi, can beperformed by standard methods of nucleophilic addition of Grignard orlithium reagents to carbonyl compounds; i.e. by reacting the RHaI withmagnesium or lithium in a suitable anhydrous solvent, such as etherand/or THF, to generate the organometallic reagent, then adding 1, togive II after usual aqueous work-up (which is normally implied in allthe reactions of Scheme 1. In general the reaction product II is amixture of the two possible C-22-epimers, here designated IIA and IIB.It is usually preferable to separate the IIA and IIB epimers which canconveniently be done by chromatography.

Nonlimiting illustrations of such compounds of formula II are given inTable 3. In this table these compounds are described as separate22-epimers IIA or IIB (preparations 1-5). The compounds IIA are formedin much higher yields than the corresponding IIB epimers, typically inthe ratio of about 95 to 5.

The alkylation or acylation of the C-22-hydroxy compounds of generalformula II to yield the corresponding compound III where R³ is C₁-C₃alkyl, aryl or aralkyl or YR⁴ can be performed by standard methods wellknown to the specialist. Illustrative, but non limiting, compounds ofthis sort are listed in Table 3.

In the alkylation reaction use is preferably made of an alkylating agentR³Z, in which Z stands for a good leaving group, such as for exampleCl—, Br—, I—, CH₃SO₃—, p—CH₃—C₆H₄—SO₃— or CF₃SO₃—; the R³Z being allowedto react with the anion of the appropriate compound II or III (R³=H),derived therefrom by means of a suitable strong base, such as analkali-metal alkoxide, alkyl alkali-metal or alkali-metal hydride. Auseful method is described in General Procedure 2, and in more detail inthe included preparations; a suitable crown ether may be added as aphase transfer agent to accelerate the alkylation process.

In the acylation reaction producing compounds III where R³=YR⁴, use mayadvantageously be made of standard acylation procedures, such asreaction of the alcohol II with an acid chloride or acid anhydride (R⁴YCl or (R⁴Y)₂O), or by forming the acylating agent in situ from thecorresponding acid R⁴YOH and a dehydrating or condensing agent, such ase.g. a carbodiimide or an assisting acid anhydride, forming anintermediary mixed anhydride.

Furthermore, the addition of a suitable base, such as a tertiary amine,may often be profitably applied during the acylation; in many casesaddition of a special heterocyclic amine like DMAP may accelerate theacylation process considerably. Examples of acylation procedures aregiven in General Procedure 3.

Table 3 also contains nonlimiting examples of photoisomerized compoundsof general formula IV along with references to the preparation of eachcompound.

It should be noted that the preparations and examples of Tables 3 and 4are illustrative only, the particular synthesis of each step and theorder in which each step is performed can be varied greatly.Furthermore, the radical R: —Q—C(R¹)(R²)(X¹) may optionally be a radicalwhich can be converted to this at any convenient later stage (or overseveral stages). Thus R in compounds II, III and IV does not necessarilyhave the same meaning along a particular synthetic sequence. Theconversion of R to —Q—C(R¹)(R²)X¹ may well involve several steps andpossibly involve a temporary protection of the sensitive triene systemof the molecule. Apart from any necessary modification of R³ or withinthe side chain (R), the conversion of III to I involves aphotoisomerisation step and a deprotection step, analogous to the stepsused in the last stages of the synthesis of other vitamin D analogues(see European patent No. 0 227 836).

TABLE 3 Intermediates of General Formulas II, III & IV Prep Comp Gen.Type No. No. Proc Radicals for formulas II, III & IV 1) 2) 3) 4) R³ Q R¹R² XR¹ IIA 01 201 1 — (CH₂)₂ Me Me OTMS IIA 02 202 1 — (CH₂)₂ Et Et OTMSIIA 03 203 1 — (CH₃)₂ Me Me OTMS IIA 04 204 1 — (CH₂)₃ Et Et OTMS IIA 05205 1 — (CH₂)₄ Et Et OTMS IIIA 06 301  2a Me (CH₂)₂ Me Me OTMS IIIA 07302  2a Et (CH₂)₂ Me Me OTMS IIIA 08 303 2 Me (CH₂)₂ Et Et OTMS IIIA 09304 2 Me (CH₂)₃ Me Me OTMS IIIA 10 305 2 Me (CH₂)₃ Et Et OTMS IIIA 11306  2a Et (CH₂)₃ Et Et OTMS IIIA 12 307 2 Me (CH₂)₄ Et Et OTMS IIIA 13308  2a Et (CH₂)₄ Et Et OTMS IVA 14 401 4 Me (CH₂)₂ Me Me OTMS IVA 15402 4 Et (CH₂)₂ Me Me OTMS IVA 16 403 4 Me (CH₂)₂ Et Et OTMS IVA 17 4044 Me (CH₂)₃ Me Me OTMS IVA 18 405 4 Me (CH₂)₃ Et Et OTMS IVA 19 406 4 Et(CH₂)₃ Et Et OTMS IVA 20 407 4 Me (CH₂)₄ Et Et OTMS IVA 21 408 4 Et(CH₂)₄ Et Et OTMS IIB 01 221 1 — (CH₂)₂ Me Me OTMS IIIA 22 310 2 Et(CH₂)₃ Me Me OTMS IIIA 23 311 2 Me (CH₂)₄ Me Me OTMS IIIA 24 312 2 Et(CH₂)₄ Me Me OTMS IIIA 25 314  2a Pr (CH₂)₂ Me Me OTMS IIIA 26 315 2 Bn(CH₂)₂ Me Me OTMS IIIB 34 321 2 Me (CH₂)₂ Me Me OTMS IVA 27 410 4 Et(CH₂)₃ Me Me OTMS IVA 28 411 4 Me (CH₂)₄ Me Me OTMS IVA 29 412 4 Et(CH₂)₄ Me Me OTMS IVA 30 414 4 Pr (CH₂)₂ Me Me OTMS IVA 31 415 4 Bn(CH₂)₂ Me Me OTMS IVA 32    416a* 4 H (CH₂)₂ Me Me OTMS IVA 33  416* 3PhOCS (CH₂)₂ Me Me OTMS IVB 35 421 4 Me (CH₂)₂ Me Me OTMS Notes to Table3: 1) Type: See Scheme 1 2) Prep No. = Preparation Number 3) Comp No. =Compound Number 4) Gen. Proc = General Procedure Number *Reversal ofsteps b) and c) of Scheme 1. Therefore, in compound 416a, R³ stands forH

Exemplified and planned Compounds I of the invention are listed in Table4, the numbered examples giving reference to illustrative methods ofsynthesis, together with spectroscopic data for these exemplifiedcompounds.

The planned Compounds, 109, 113, 117-120 and 122-135 are made in asequence of synthetic steps which is analogous to the sequence used forthe preparations of the exemplified Compounds, 101-108, 110-112, 114-116and 121, Examples 1-15; see Scheme 1, and the notes to Scheme 1.

In the following, R⁴, R⁵, Hal, Z and Y have the above meanings, and Q,R¹, R² and R³ have same meanings as they have in Table 4, for thecorresponding planned compounds of formula 1. Compound 1 and theappropriate Compound V, HaI—Q—CR¹R²—OR⁵ (OR⁵ being replaced by H in thecase of the synthesis of Compound 131), are reacted, according toGeneral Procedure 1 (GP 1), to give the corresponding compound offormula II.

The compound of formula II and the appropriate R³Z are reacted,according to GP 2, to give the corresponding compound of formula III,except for the sequence leading to Compounds 134 and 135; in which casesthe appropriate Compound II (Compound 201) is acylated, according to GP3, with the appropriate acylating reagent R⁴YCl or (R⁴Y)₂O to give thecorresponding compound of formula III.

The compound of formula III is photo-isomerized, according to GP 4, togive the corresponding compound of formula IV. As the last step, thecompound of formula IV is deprotected, according to either GP 5, GP 6 orGP 7, to give the Compound I in question.

TABLE 4 Exemplified and Planned Compounds of General Formula I Exam CompIsom Gen. No. No. C-22 Proc Radicals of the general formula I 1) 2) 3)4) R³ Q R¹ R² X  1 101 A 5 Me (CH₂)₂ Me Me OH  2 102 A 5 Et (CH₂)₂ Me MeOH  3 103 A 5 Me (CH₂)₂ Et Et OH  4 104 A 5 Me (CH₂)₃ Me Me OH  5 105 A5 Me (CH₂)₃ Et Et OH  6 106 A 6 Et (CH₂)₃ Et Et OH  7 107 A 6 Me (CH₂)₄Et Et OH  8 108 A 6 Et (CH₂)₄ Et Et OH 109 A 5-7 Et (CH₂)₂ Et Et OH  9110 A 5 Et (CH₂)₃ Me Me OH 10 111 A 5 Me (CH₂)₄ Me Me OH 11 112 A 5 Et(CH₂)₄ Me Me OH 113 A 5-7 Me CH₂ Me Me OH 12 114 A 5 Pr (CH₂)₂ Me Me OH13 115 A 5 Bn (CH₂)₂ Me Me OH 14 116 A 6 PhOCS (CH₂)₂ Me Me OH 117 A 5-7Me (CH₂)₂ CF₃ CF₃ OH 118 A 5-7 Et (CH₂)₂ CF₃ CF₃ OH 119 A 5-7 Me (CH₂)₂C₂F₅ C₂F₅ OH 120 A 5-7 Et (CH₂)₂ C₂F₅ C₂F₅ OH 15 121 B 5 Me (CH₂)₂ Me MeOH 122 B 5-7 Et (CH₂)₂ Me Me OH 123 A 5-7 Me (CH₂)₃ CF₃ CF₃ OH 124 A 5-7Et (CH₂)₃ CF₃ CF₃ OH 125 A 5-7 Me CH₂CF₂ Me Me OH 126 A 5-7 Et CH₂CF₂ MeMe OH 127 A 5-7 Me CH₂CF₂ CF₃ CF₃ OH 128 A 5-7 Et CH₂CF₂ CF₃ CF₃ OH 129* A 5-7 Me (CH₂)₂ Me CF₃ OH  130# A 5-7 Me (CH₂)₂ CF₃ Me OH 131 A5-7 Me (CH₂)₂ Me Me H 132 A 5-7 Me (CH₂)₂ —(CH₂)₄— OH 133 A 5-7 Me(CH₂)₂ —(CH₂)₅— OH 134 A 6 PhSCO (CH₂)₂ Me Me OH 135 A 6 PhSCS (CH₂)₂ MeMe OH Notes to Table 4 1) Exam No. = Example Number 2) Comp No. =Compound Number 3) Isom C-22 = Isomer at carbon 22 of the side chain 4)Gen. Proc = General Procedure Number *R-configuration of the carbonbearing R¹ and R² #S-configuration of the carbon bearing R¹ and R²

The present compounds are intended for use in pharmaceuticalcompositions which are useful in the local or systemic treatment ofhuman and veterinary disorders as described above.

The present compounds may be used in combination with otherpharmaceuticals or treatment modalities. In the treatment of psoriasisthe present compounds may be used in combination with e.g. steroids orwith other treatments e.g. light- or UV-light-treatment or the combinedPUVA-treatment. In the treatment of cancer the present compounds may beused in combination with other anti-cancer drugs or anti-cancertreatments, such as radiation treatment. In the prevention of graftrejection and graft versus host reaction, or in the treatment ofauto-immune diseases, the present compounds may advantageously be usedin combination with other immunosuppressive/immunoregulating drugs ortreatments, e.g. with cyclosporin A.

The amount required of a compound of formula I (hereinafter referred toas the active ingredient) for therapeutic effect will, of course, varyboth with the particular compound, the route of administration and themammal under treatment. The compounds of the invention can beadministered by the parenteral, intra-articular, enteral or topicalroutes. They are well absorbed when given enterally and this is thepreferred route of administration in the treatment of systemicdisorders. In the treatment of dermatological disorders like psoriasisor eye diseases topical or enteral forms are preferred.

While it is possible for an active ingredient to be administered aloneas the raw chemical, it is preferable to present it as a pharmaceuticalformulation. Conveniently, the active ingredient comprises from 0.1 ppmto 0.10% by weight of the formulation.

The formulations, both for veterinary and for human medical use, of thepresent invention thus comprise an active ingredient in association witha pharmaceutically acceptable carrier therefore and optionally othertherapeutic ingredient(s). The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationsand not deleterious to the recipient thereof.

The formulations include e.g. those in a form suitable for oral,ophthalmic, rectal, parenteral (including subcutaneous, intramuscularand intravenous), transdermal, intra-articular and topical, nasal orbuccal administration.

By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient, and which may be readilyhandled and packed, remaining as a physically and chemically stable unitdose comprising either the active material as such or a mixture of itwith solid or liquid pharmaceutical diluents or carriers.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active ingredient into association with a liquidcarrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion. Theactive ingredient may also be administered in the form of a bolus,electuary or paste.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and a carrier, or in theform of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.Transdermal formulations may be in the form of a plaster.

Formulations suitable for intra-articular or ophthalmic administrationmay be in the form of a sterile aqueous preparation of the activeingredient which may be in microcrystalline form, for example, in theform of an aqueous microcrystalline suspension. Liposomal formulationsor biodegradable polymer systems may also be used to present the activeingredient for both intra-articular and ophthalmic administration.

Formulations suitable for topical or ophthalmic administration includeliquid or semi-liquid preparations such as liniments, lotions, gels,applicants, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes; or solutions or suspensions such as drops.

Formulations suitable for administration to the nose or buccal cavityinclude powder, self-propelling and spray formulations, such as aerosolsand atomizers.

In addition to the aforementioned ingredients, the formulations of thisinvention may include one or more additional ingredients, such asdiluents, binders, preservatives etc.

The compositions may further contain other therapeutically activecompounds usually applied in the treatment of the above mentionedpathological conditions, such as other immunosuppressants in thetreatment of immunological diseases, or steroids in the treatment ofdermatological diseases.

The present invention further concerns a method for treating patientssuffering from one of the above pathological conditions, said methodconsisting of administering to a patient in need of treatment aneffective amount of one or more compounds of formula 1, alone or incombination with one or more other therapeutically active compoundsusually applied in the treatment of said pathological conditions. Thetreatment with the present compounds and/or with further therapeuticallyactive compounds may be simultaneous or with intervals.

In the systemic treatment daily doses of from 0.001-2 μg per kilogrambodyweight, preferably from 0.002-0.3 μg/kg of mammal bodyweight, forexample 0.003-0.3 pg/kg of a compound of formula I are administered,typically corresponding to a daily dose for an adult human of from 0.2to 25 μg. In the topical treatment of dermatological disorders,ointments, creams or lotions containing from 0.1-500 μg/g, andpreferably from 0.1-100 μg/g, of a compound of formula I areadministered. For topical use in ophthalmology ointments, drops or gelscontaining from 0.1-500 pg/g, and preferably from 0.1-100 μg/g, of-acompound of formula I are administered. The oral compositions areformulated, preferably as tablets, capsules, or drops, containing from0.05-50 μg, preferably from 0.1-25 μg, of a compound of formula 1, perdosage unit.

The invention will now be further described in the following GeneralProcedures, Preparations and Examples:

General Procedures, Preparations and Examples General

The exemplified compounds I are listed in Table 4.

For ¹H NMR (300 MHz) and ¹³C NMR (75.6 MHz) spectra chemical shiftvalues (δ) are quoted, unless otherwise specified, fordeuteriochloroform solutions relative to internal tetramethylsilane(δ=0.00) or chloroform (δ=7.25 for 1H NMR) or deuteriochloroform(δ=76.81 for ¹³C NMR). The value for a multiplet, either defined(doublet (d), triplet (t), quartet (q)) or not (m) at the approximatemid point is given unless a range is quoted (s=singlet, b=broad).

Ether is diethyl ether, and was dried over sodium. THF was dried oversodium-benzophenone. Petroleum ether refers to the pentane fraction.Reactions were run at room temperature unless otherwise noted. Thework-up procedure referred to involves dilution with the'specifiedsolvent (otherwise the organic reaction solvent), extraction with waterand then brine, drying over anhydrous MgSO₄, and concentration in vacuoto give a residue. Chromatography was performed on silica gel.

General Procedures

General Procedure 1: Reaction of Compound 1 with Grignard reagents,RMgHaI, derived from side chain building blocks V (RHaI). to giveCompounds IIA and IIB (Scheme 1. Table 3) (Preparations 01-05)

To 0.12 g magnesium turnings (Grignard quality) in a dry flask wasadded, dropwise with stirring and in an argon atmosphere, a solution ofthe appropriate compound V (5.0 mmol) in a 1:1 mixture of dry THF anddry ether (6 ml). Stirring was continued under heating to reflux for 45minutes. The resulting Grignard reagent was again cooled to 20° C., anda solution of the aldehyde, compound 1 (0.57 g; 1 mmole) in a 1:1mixture of dry THF and dry ether (2 ml) was added dropwise, during 5minutes, followed by stirring at 20° C. for 30 minutes. The reactionmixture was poured into a 10% aqueous solution of ammonium chloride andworked up (ether) to yield a crude product containing compounds IIA andIIB which were separated and purified by chromatography (mixture ofether and pet.ether as eluant) to yield the title compounds of thepreparation. (If necessary, repeated chromatography of selectedfractions were performed, possibly using mixtures of pet.ether,dichlormethane and ethylacetate, e.g. a 80:20:2 mixture, as eluant.Conveniently the purification/separation may take place in the form ofPLC or using a preparative HPLC apparatus).

General Procedure 2: Alkylation of C-22-hydroxy-compounds of type II orIII to the corresponding compound III where R³=C₁-C₁₀ hydrocarbyl(Scheme 1, Table 3) (Preparations 06-13 and 22-23)

To a solution of the appropriate compound II or III (0.25 mmol) in dryTHF (4 ml) was added, while stirring at 20° C. under argon, a 20%suspension of potassium hydride in mineral oil (0.1 ml) followed by analkylating agent, R³Z (0.75 mmol), and the mixture was stirred for 5minutes, after which 18-Crown-6 (0.07 g) was added. Stirring at 20° C.was continued for 1.5 hours, after which the reaction mixture was workedup (ether). The crude product was purified by chromatography (mixture ofether and pet.ether as eluant) to yield the title compound of thepreparation.

Variation: General Procedure 2a

The procedure of General Procedure 2 was followed, except that afterstirring of the reaction mixture for 1.5 hours as described, anotherportion of potassium hydride (20% in mineral oil) (0.1 ml), ofalkylating agent R³Z (0.75 mmol), and of 18-crown-6 (0.07 g) was added,and the mixture was stirred for another 1.5 hours.

General Procedure 3: Acylation of C-22-hydroxy-compounds of type II orIII to the corresponding compound III where R³=YR⁴ (Scheme 1 Table 3)(Preparation 33)

To a solution of the appropriate compound II or III (0.25 mmol) in asuitable dry solvent, e.g. dichloromethane, was added, while stirring at20° C. under argon, an acylating reagent (R⁴YCl, (R⁴Y)₂O or R⁴YOH),preferably accompanied by one or two suitable bases, such astriethylamine, pyridine and/or DMAP. In the case where an acid, R⁴YOH,was used, the addition of a dehydrating or condensing agent such as e.g.dicyclohexyl carbodiimide was desirable. The reaction mixture was thenstirred at a suitable temperature (from room temperature up to theboiling point of the solvent) for a sufficient time (typically for 1 to4 hours). After a suitable work-up the crude product was purified bychromatography to yield the title compound of the preparation.

General Procedure 4: Isomerization of Compounds III to the correspondingcompound IV (Scheme 1, Table 3) (Preparations 14-21 and 27-35)

A solution of the appropriate compound III (0.3 mmol), anthracene (120mg) and triethylamine (0.05 ml) in dichloromethane (20 ml) under argonin a Pyrex flask was irradiated with UV-light from a high pressureultraviolet lamp, type TQ760Z2 (Hanau) at about 1I0° C. for 20 minutesunder stirring. The reaction mixture was concentrated in vacuo andtreated with pet.ether (2×5 ml). After filtering the filtrate wasconcentrated in vacuo and purified by chromatography (mixture of etherand pet.ether as eluant) to yield the title compound of the preparation.General Procedure 5: Deprotection of Compounds IV to the correspondingCompounds I by treatment with tetra-n-butyl-ammoniumfluoride (Scheme 1.Table 4) (Examples 1-5, 9-11, 12-13 and 15)

To a solution of the appropriate compound IV (0.16 mmol) in THF (5 ml)was added a solution of tetra-n-butylammonium fluoride (300 mg) in THF(5 ml) while stirring at 60° C. under argon. Stirring was continued forone hour at 60° C., and the reaction mixture was worked up (ethylacetate with an additional extraction with aqueous sodium hydrogencarbonate). The residue after evaporation was purified by chromatography(50% to 0% pet.ether in ethyl acetate as eluant) to yield the titlecompound of the example.

General Procedure 6: Deprotection of Compounds IV to the correspondingCompound I by treatment with HF (Scheme 1, Table 4) (Examples 6-8 and14)

To a solution of the appropriate compound IV (0.07 mmol) in ethylacetate (0.2 ml) was added acetonitrile (2 ml) followed by a 5% solutionof hydrofluoric acid in acetonitrile:water, 7:1 (1.2 ml) under argon andwith stirring. Stirring was continued for 45 minutes at 20° C. Saturatedaqueous sodium bicarbonate solution (10 ml) was added, and the reactionmixture was worked up (ethyl acetate) The residue was purified bychromatography (50% to 0% pet.ether in ethyl acetate as eluant) to yieldthe title compound of the example.

General Procedure 7: Deprotection of compounds IV to the corresponddingCompounds I by treatment with tetra-n-butylammoniumfluoride followed bypyridine-p-toluenesulfonate² (Scheme 1, Table 4)

² or in the reverse order

To a solution of the appropriate compound IV (0.16 mmol) in THF (5 ml)was added a solution of tetra-n-butylammonium fluoride (300 mg) in THF(5 ml) while stirring at 60° C. under argon. Stirring was continued forone hour at 60° C., and the reaction mixture was worked up (ethylacetate with an additional extraction with aqueous sodium hydrogencarbonate). The residue after evaporation was purified by chromatography(50% to 0% pet.ether in ethyl acetate as eluant) and then dissolved inabsolute ethyl alcohol (2 ml). PPTS (2 mg) was added, and the mixturewas stirred for one hour at 50° C. under argon. After work-up (ethylacetate with an additional aqueous sodium bicarbonate extraction) theresidual crude product was purified by chromatography (50% to 0%pet.ether in ethyl acetate as eluant) to yield the title compound of theexample.

Preparations

Preparation 01: Compounds 201 and 221

Method: General Procedure 1.

Starting material V: Compound 501

Chromatography eluant: 0% to 5% ether in pet.ether.

¹H NMR for 201: δ=0.06 (m, 12H), 0.11 (s, 9H), 0.54 (s, 3H), 0.85 (d,3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.23 (s, 6H), 1.20-2.15 (m, 19H), 2.31(bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 380 (m, 1H), 4.21 (m, 1H), 4.52(m, 1H), 4.94 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H), 6.45 (d, 1H).

¹³C NMR for 221: 153.4, 142.8, 135.3, 121.5, 116.3, 106.5, 74.1, 73.8,70.1, 67.0, 56.1, 52.7, 45.7, 43.8, 41.8, 41.2, 39.8, 36.4, 29.9, 29.5,28.7, 26.7, 25.7, 25.6, 25.2, 23.3, 21.9, 18.1, 17.9, 12.4, 12.2, 2.3,−5.0, −5.1,-

Preparation 02: Compound 202

Method: General Procedure 1.

Starting material V: Compound 502

Chromatography eluant: 5% ether in pet.ether.

¹H NMR: δ=0.05 (m, 12H), 0.11 (s, 9H), 0.54 (s, 3H), 0.82 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.15-2.15 (m, 23H), 2.31 (m, 1H),2.55 (dd, 1H), 2.88 (m, 1H), 3.77 (m, 1H), 4.21 (m, 1H), 4.52 (m, 1H),4.93 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H), 6.45 (d. 1H).

Preparation 03: Compound 203

Method: General Procedure 1.

Starting material V: Compound 503

Chromatography eluant: 5% ether in pet.ether.

¹H NMR: δ32 0.06 (m, 12H), 0.10 (s, 9H), 0.55 (s, 3H), 0.83 (d, 3H),0.86 (s, 9H), 0.89 (s, 9H), 1.20 (s, 6H), 1.15-2.15 (m, 21H), 2.31 (bd,1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.85 (m, 1H), 4.21 (m, 1H), 4.52 (m,1H), 4.94 (m, 1H), 4.98 (m, 1H), 5.83 (d, 1H), 6.45 (d, 1H).

Preparation 04: Compound 204

Method: General Procedure 1.

Starting material V: Compound 504

Chromatography eluant: 10% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.09 (s, 9H), 0.55 (s, 3H), 0.81 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.45 (q, 4H), 1.15-2.15 (m, 21H),2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.85 (m, 1H), 4.22 (m, 1H),4.53 (m, 1H), 4.94 (m, 1H), 4.98 (m, 1H), 5.83 (d, 1H), 6.45 (d, 1H).

Preparation 05: Compound 205

Method: General Procedure 1.

Starting material V: Compound 505

Chromatography eluant: 0% to 5% ether in pet.ether.

¹H NMR: δ=0.05 (m, 12H), 0.08 (s, 9H), 0.54 (s, 3H), 0.79 (t, 6H), 0.83(d, 3H), 0.85 (s, 9H), 0.89 (s, 9H), 1.10-2.10 (m, 27H), 2.31 (bd, 1H),2.55 (dd, 1H), 2.88 (m, 1H), 3.84 (m, 1H), 4.21 (m, 1H), 4.53 (m, 1H),4.94 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H), 6.45 (d, 1H).

Preparation 06: Compound 301

Method: General Procedure 2a.

Alkylating agent R³Z: Methyl iodide

Starting material II: Compound 201

Chromatography eluant: 0% to 20% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.10 (s, 9H), 0.51 (s, 3H), 0.83 (d, 3H), 0.86(s, 9H), 0.89 (s, 9H), 1.20 (s, 3H), 1.21 (s, 3H), 1.25-2.10 (m, 18H),2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.18 (m, 1H), 3.33 (s, 3H),4.21 (m, 1H), 4.52 (m, 1H), 4.94 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H),6.45 (d, 1H).

Preparation 07: Compound 302

Method: General Procedure 2a.

Starting material II: Compound 201

Alkylating agent R³Z: Ethyl iodide

Chromatography eluant: 0% to 3% ether in pet.ether.

¹H NMR: δ=0.05 (m, 12H), 0.09 (s, 9H), 0.50 (s, 3H), 0.84 (d, 3H), 0.86(s, 9H), 0.89 (s, 9H), 1.17 (t, 3H), 1.20 (s, 3H), 1.21 (s, 3H),1.10-2.10 (m, 18H), 2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.20 (m,1H), 3.47 (m, 2H), 4.22 (m, 1H), 4.52 (m, 1H), 4.93 (m, 1H), 4.98 (m,1H), 5.81 (d, 1H), 6.45 (d, 1H).

Preparation 08: Compound 303

Method: General Procedure 2.

Starting material II: Compound 202

Alkylating agent R³Z: Methyl iodide

Chromatography eluant: 0% to 20% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.10 (s, 9H), 0.52 (s, 3H), 0.82 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.20-2.15 (m, 22H), 2.31 (bd, 1H),2.55 (dd, 1H), 2.88 (m, 1H), 3.18 (m, 1H), 3.33 (s, 3H), 4.21 (m, 1H),4.53 (m, 1H), 4.93 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H), 6.46 (d, 1H).

Preparation 09: Compound 304

Method: General Procedure 2.

Starting material II: Compound 203

Alkylating agent R³Z: Methyl iodide

Chromatography eluant: 0% to 2% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.09 (s, 9H), 0.52 (s, 3H), 0.83 (d, 3H), 0.86(s, 9H), 0.89 (s, 9H), 1.19 (s, 3H), 1.20 (s, 3H), 1.10-2.15 (m, 20H),2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1 (H), 3.22 (m, 1.H), 3.33 (s, 3(H), 4.21 (m, 1H), 4.52 (m, 1H), 4.93 (m, 1H), 4.98 (m, 1H), 5.82 (d,1H), 6.45 (d, 1H).

Preparation 10: Compound 305

Method: General Procedure 2.

Starting material II: Compound 204

Alkylating agent R³Z: Methyl iodide

Chromatography eluant: 0% to 20% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.10 (s, 9H), 0.52 (s, 3H), 0.81 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.15-2.10 (m, 24H), 2.31 (bd, 1H),2.55 (dd, 1H), 2.88 (m, 1H), 3.21 (m, 1H), 3.33 (s, 3H), 4.21 (m, 1H),4.52 30 (m, 1H), 4.93 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H), 6.45 (d, 1H).

Preparation II: Compound 306

Method: General Procedure 2a.

Starting material II: Compound 204

Alkylating agent R³Z: Ethyl bromide

Chromatography eluant: 0% to 20% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.08 (s, 9H), 0.50 (s, 3H), 0.81 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.17 (t, 3H), 1.10-2.10 (m, 24H),2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.24 (m, 1H), 3.47 (m, 2H),4.21 (m, 1H), 4.53 (m, 1H), 4.93 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H),6.45 (d, 1H).

Preparation 12: Compound 307

Method: General Procedure 2.

Starting material II: Compound 205

Alkylating agent R³Z: Methyl iodide Chromatography eluant: 0% to 20%ether in pet.ether.

¹ H NMR: δ=0.06 (m, 12H), 0.09 (s, 9H), 0.51 (s, 3H), 0.80 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.44 (q 4H), 1.15-2.10 (m, 22H),2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.22 (m, 1H), 3.33 (s, 3H),4.21 (m, 1H), 4.53 (m, 1H), 4.93 (m, 1H), 4.98 (m, 1H), 5.82 (d, 1H),6.45 (d, 1H).

Preparation 13: Compound 308

Method: General Procedure 2a.

Starting material II: Compound 205

Alkylating agent R³Z: Ethyl bromide

Chromatography eluant: 0% to 20% ether in pet.ether.

¹H NMR: δ=0.05 (m, 12H), 0.09-(s, 9H), 0.50 (s, 3H), 0.80 (t, 6H), 0.83(d, 3H), 0.86 (s, 9H), 0.89 (s, 9H), 1.17 (t, 3H), 1.44 (q, 4H),1.15-2.10 (m, 22H), 2.31 (bd, 1H), 2.55 (dd, 1H), 2.88 (m, 1H), 3.22 (m,1H), 3.47 (m, 2H), 4.21 (m, 1H), 4.53 (m, 1H), 4.93 (m, 1H), 4.98 (m,1H), 5.82 (d, 1H), 6.45 (d, 1H).

Preparation 14: Compound 401

Method: General Procedure 4.

Starting material III: Compound 301

Chromatography eluant: 5% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.11 (s, 9H), 0.50 (s, 3H), 0.82 (d, 3H), 0.87(s, 18H), 1.20 (s, 3H), 1.21 (s, 3H), 1.15-2.10 (m, 18H), 2.21 (dd, 1H),2.44 (dd, 1H), 2.82 (m, 1H), 3.18 (m, 1H), 3.32 (s, 3H), 4.18 (m, 1H),4.37 (m, 1H), 4.86 (m, 1H), 5.17 (m, 1H), 6.01 (d, 1H), 6.23 (d, 1H).

Preparation 15: Compound 402

Method: General Procedure 4.

Starting material III: Compound 302

Chromatography eluant: 2.5% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.10 (s, 9H), 0.49 (s, 3H), 0.83 (d, 3H), 0.88(s, 18H), 1.17 (t, 3H), 1.20 (s, 3H), 1.21 (s, 3H), 1.10-2.10 (m, 18H),2.21 (dd, 1H), 2.44 (dd, 1H), 2.83 (m, 1H), 3.20 (m, 1H), 3.47 (m, 2H),4.18 (m, 1H), 4.37 (m, 1H), 4.86 (m, 1H), 5.17 (m, 1H), 6.01 (d, 1H),6.23 (d, 1H).

Preparation 16: Compound 403

Method: General Procedure 4.

Starting material III: Compound 303

Chromatography eluant: 0% to 2% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.10 (s, 9H), 0.50 (s, 3H), 0.82 (m, 9H), 0.87(s, 9H), 0.88 (s, 9H), 1.20-2.10 (m, 22H), 2.21 (dd, 1H), 2.44 (dd, 1H),2.82 (m, 1H), 3.18 (m, 1H), 3.32 (s, 3H), 4.18 (m, 1H), 4.38 (m, 1H),4.86 (m, 1H), 5.18 (m, 1H), 6.01 (d, 1H), 6.23 (d, 1H).

Preparation 17: Compound 404

Method: General Procedure 4.

Starting material III: Compound 304

Chromatography eluant: 2% ether in pet.ether.

¹H NMR: δ=0.05 (m, 12H), 0.09 (s, 9H), 0.50 (s, 3H), 0.81 (d, 3H), 0.86(s, 18H), 1.19 (s, 3H), 1.20 (s, 3H), 1.15-2.07 (m, 20H), 2.21 (dd, 1H),2.44 (dd, 1H), 2.82 (m, 1H), 3.22 (m, 1H), 3.32 (s, 3H), 4.18 (m, 1H),4.37 (m, 1H), 4.86 (m, 1H), 5.17 (m, 1H), 6.00 (d, 1H), 6.23 (d, 1H).

Preparation 18: Compound 405

Method: General Procedure 4.

Starting material III: Compound 305

Chromatography eluant: 0% to 3% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.09 (s, 9H), 0.51 (s, 3H), 0.81 (m, 9H), 0.88(s, 18H), 1.15-2.10 (m, 24H), 2.21 (dd, 1H), 2.43 (dd, 1H), 2.82 (m,1H), 3.21 (m, 1H), 3.32 (s, 3H), 4.18 (m, 1H), 3.37 (m, 1H), 4.86 (m,1H), 5.17 (m, 1H), 6.01 (d, 1H), 6.23 (d, 1H).

Preparation 19: Compound 406

Method: General Procedure 4.

Starting material III: Compound 306

Chromatography eluant: 0% to 2% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.08 (s, 9H), 0.49 (s, 3H), 0.81 (t, 6H), 0.82(d, 3H), 0.87 (s, 18H), 1.17 (t, 3H), 1.10-2.10 (m, 24H), 2.21 (dd, 1H),2.44 (dd, 1H), 2.82 (m, 1H), 3.23 (m, 1H), 3.47 (q, 2H), 4.18 (m, 1H),4.37 (m, 1H), 4.86 (m, 1H), 5.18 (m, 1H), 6.01 (d, 1H), 6.23 (d, 1H).

Preparation 20: Compound 407

Method: General Procedure 4.

Starting material III: Compound 307

Chromatography eluant: 0% to 2% ether in pet.ether.

¹H NMR: δ=0.06 (m, 12H), 0.09 (s, 9H), 0.50 (s, 3H), 0.80 (t, 6H), 0.81(d, 3H), 0.87 (s, 18H), 1.44 (q, 4H), 1.10-2.10 (m, 22H), 2.21 (dd, 1H),2.44 (dd, 1H), 2.83 (m, 1H), 3.21 (m, 1H), 3.32 (s, 3H), 4.18 (m, 1H),4.36 (m, 1H), 4.86 (m, 1H), 5.17 (m, 1H), 6.01 (d, 1H), 6.23 (d, 1H).

Preparation 21: Compound 408

Method: General Procedure 4.

Starting material III: Compound 308

Chromatography eluant: 0% to 2% ether in pet.ether.

¹H NMR: δ 0.06 (m, 12H), 0.09 (s, 9H), 0.49 (s, 3H), 0.79 (t, 6H), 0.80(d, 3H), 0.87 (s, 18H), 1.17 (t, 3H), 1.44 (q, 4H), 1.07-2.10 (m, 22H),2.21 (dd, 1H), 2.44 (dd, 1H), 2.83 (m, 1H), 3.22 (m, 1H), 3.47 (m, 2H),4.18 (m, 1H), 4.36 (m, 1H), 4.86 (m, 1H), 5.17 (m, ₁H), 6.01 (d, 1H),6.23 (d, 1H).

Preparation 22: Compound 310

Method: General Procedure 2.

Starting material II: Compound 203

Alkylating agent R³Z: Ethyl bromide

Chromatography eluant: 0% to 5% ether in pet.ether.

¹³C NMR: 153.4, 143.3, 135.1, 121.6, 116.1, 106.4, 82.6, 73.8, 70.1,67.0, 64.2, 55.6, 50.7,46.1, 44.9, 43.8, 39.2, 36.4, 35.0, 31.1, 29.7,29.6, 28.9, 25.7, 25.6, 25.0, 23.2, 22.1, 21.1, 18.1, 17.9, 15.6, 12.8,12.7, 2.4, −4.9, −5.0, −5.1, −5.1.

Preparation 23: Compound 311

Method: General Procedure 2.

Starting material II: Compound 114j of WO91/00271

Alkylating agent R³Z: Methyl iodide

Chromatography eluant: 0% to 20% ether in pet.ether.

¹³C NMR: 153.4, 143.2, 135.1, 121.6, 116.2, 106.4, 83.4, 73.8, 70.1,67.0, 56.4, 55.8, 51.2, 45.9, 44.8, 43.8, 39.6, 36.4, 35.4, 30.2, 29.7,29.6, 28.8, 26.7, 25.8, 25.7, 25.6, 24.5, 23.3, 22.0, 18.1, 17.9, 12.6,12.3, 2.4, −4.9, −5.0, −5.1, −5.1

Preparation 24: Compound 312

Method: General Procedure 2.

Starting material II: Compound 114j of WO91/00271

Alkylating agent R³Z: Ethyl bromide

Chromatography eluant: 0% to 20% ether in pet.ether.

¹³C NMR: 153.4, 143.3, 135.1, 121.6, 116.1, 106.4, 82.5, 73.8, 70.1,67.0, 64.2, 55.7, 50.8, 46.1, 44.8, 43.8, 39.2, 36.4, 35.1, 30.8, 29.7,29.6, 28.9, 26.9, 25.7, 25.6, 25.1, 24.5, 23.3, 22.1, 18.1, 17.9, 15.6,12.8, 12.6, 2.4, 4.9, −5.0, −5.1, −5.1

Preparation 25: compound 314

Method: General Procedure 2a.

Starting material II: Compound 201

Alkylating agent R³Z: n-Propyl bromide

Chromatography eluant: 0% to 5% ether in pet.ether.

¹³C NMR: 153.5, 143.3, 135.1, 121.6, 116.1, 106.4, 83.0, 73.8, 70.6,70.1, 67.0, 55.7, 50.9, 46.1, 43.8, 41.2, 39.4, 36.4, 35.3, 29.8, 29.6,28.9, 25.7, 25.6, 25.3, 25.2, 23.4, 23.3, 22.1, 18.1, 17.9, 12.6, 10.7,2.4, 4.9, −5.0, −5.1, −5.1

Preparation 26: Compound 315

Method: General Procedure 2.

Starting material II: Compound 201

Alkylating agent R³Z: Benzyl bromide

Chromatography eluant: 0% to 5% ether in pet.ether.

¹³C NMR: 153.4, 143.2, 138.1, 135.1, 128.0, 127.4, 127.0, 121.6, 116.2,106.4, 82.7, 73.7, 70.4, 70.1, 67.0, 55.7, 51.1, 46.0, 43.8, 41.2, 39.5,36.4, 35.5, 29.8, 29.6, 28.8, 25.7, 25.6, 25.5, 25.2, 23.2, 22.0, 18.1,17.9, 12.6, 12.5, 2.4, −4.9, −5.0, −5.1, −5.1

Preparation 27: Compound 410

Method: General Procedure 4.

Starting material III: Compound 310

Chromatography eluant: 0% to 5% ether in pet.ether.

¹³C NMR: 148.1, 140.9, 134.7, 123.0, 117.6, 111.0, 82.7, 73.8, 71.8,67.3, 64.2, 55.5, 50.7, 46.0, 45.8, 44.9, 44.6, 39.2, 35.0, 31.2, 29.7,29.6, 28.8, 25.7, 25.6, 25.0, 23.2, 22.0, 21.1, 18.1, 18.0, 15.6, 12.9,12.6, 2.4, 4.9, −5.0, −5.3

Preparation 28: Compound 411

Method: General Procedure 4.

Starting material III: Compound 311

Chromatography eluant: 0% to 5% ether in pet.ether.

¹³C NMR: 148.1, 140.8, 134.8, 123.0, 117.6, 111.0, 83.4, 73.7, 71.8,67.3, 56.4, 55.6, 51.2, 45.8, 45.7, 44.7, 44.6, 39.7, 35.4, 30.1, 29.7,29.6, 28.7, 26.7, 25.8, 25.7, 25.6, 24.5, 23.3, 21.9, 18.0, 17.9, 12.5,12.3, 2.4, −4.9, −5.0, −5.3

Preparation 29: Compound 412

Method: General Procedure 4.

Starting material III: Compound 312

Chromatography eluant: 0% to 5% ether in pet.ether.

¹³C NMR: 148.1, 140.9, 134.7, 123.0, 117.6, 111.0, 82.6, 73.8, 71.8,67.3, 64.2, 55.5, 50.7, 46.0, 45.8, 44.8, 44.6, 39.3, 35.1, 30.8, 29.7,29.6, 28.8, 26.9, 25.7, 25.7, 25.6, 25.1, 24.5, 23.2, 22.0, 18.1, 15.6,12.8, 12.6, 2.4, −4.9, −5.0, −5.3

Preparation 30: Compound 414

Method: General Procedure 4.

Starting material III: Compound 314

Chromatography eluant: 0% to 1% ether in pet.ether.

¹³C NMR: 148.1, 140.9, 134.7, 123.0,117.6, 111.0, 83.0, 73.8, 71.8,70.6, 67.3, 55.6, 50.9, 45.9, 45.8, 44.6, 41.2, 41.2, 39.5, 35.3, 29.8,29.6, 27.5, 25.7, 25.6, 25.3, 25.2, 23.4, 22.4, 20.2, 19.2, 14.1, 12.6,10.7, 2.4, −4.9, −5.0, −5.3

Preparation 31: Compound 415

Method: General Procedure 4.

Starting material III: Compound 315

Chromatography eluant: 001 to 1% ether in pet.ether.

¹³C NMR: 148.1, 140.8, 139.3, 134.8, 128.0, 127.2, 127.0, 123.0, 117.6,111.0, 82.7, 73.7, 71.8, 70.4, 67.3, 55.6, 51.1, 45.9, 45.8, 44.6, 41.2,39.5, 35.5, 29.8, 29.6, 28.7, 25.7, 25.6, 25.5, 25.2, 23.2, 21.9, 18.1,18.0, 12.6, 12.5, 2.4, −4.8, −4.9, −5.0, −5.3

Preparation 32: Compound 416a

Method: General Procedure 4.

Starting material II: Compound 201

Chromatography eluant: 0% to 10% ether in pet.ether.

¹³C NMR: 148.1, 140.6, 134.9, 122.9, 117.8, 111.0, 73.9, 73.0, 71.9,67.3, 56.0, 52.3, 45.8, 45.3, 44.6, 41.6, 40.0, 39.8, 29.8, 29.7, 29.6,28.7, 27.2, 25.7, 25.6, 23.2, 21.8, 18.0, 17.9, 12.2, 11.2, 2.4, −4.9,−5.0, −5.3

Preparation 33: Compound 416

Method: General Procedure 3.

Starting material IV: Compound 416a

Acylating reagent: Phenyl chlorothionoformate (125 mg)

Bases: Pyridine (0.2 ml); 4-Dimethylaminopyridine (115 mg)

Solvent: Dichloromethane (4 ml).

Reaction temperature: 20° C.

Reaction time: 7 hours.

Chromatography eluant: 5% ether in pet.ether.

¹³C NMR: 194.3, 153.1, 148.2, 140.4, 135.0, 129.2, 126.2, 122.9, 121.9,117.8, 111.0, 89.2, 73.3, 71.8, 67.3, 55.5, 51.3, 45.8, 45.6, 44.6,40.2, 39.1, 34.9, 29.9, 29.4, 28.7, 25.7, 25.6, 25.4, 24.3, 23.1, 21.9,18.1, 18.0, 12.6, 12.4, 2.4, −4.9, −5.0, −5.3

Preparation 34: Compound 321

Method: General Procedure 2.

Alkylating agent R³ Z: Methyl iodide

Starting material II: Compound 221

Reaction time: 15 minutes

Chromatography eluant: 15% ether in pet.ether: PLC.

¹H NMR: δ=6.45 (d, 1H), 5.83 (d, 1H), 4.98 (m, 1H), 4.93 (m, 1H), 4.53(m, 1H), 4.22 (m, 1H), 3.30 (s, 3H), 3.24 (m, 1H), 2.88 (m, 1H), 2.55(dd, 1H), 2.31 (m, 1H), 2.08-1.15 (m, 18H), 1.22 (s, 3H), 1.19 (s, 3H),0.89 (s, 9H), 0.86 (s, 9H), 0.81 (d, 3H), 0.58 (s, 3H), 0.10 (s, 9H),0.05 (m, 12H)

Preparation 35: Compound 421

Method: General Procedure 4.

Starting material III: Compound 321

Chromatography eluant: 0% to 1% ether in pet.ether.

¹H NMR: δ=6.24 (d, 1H), 6.03 (d, 1H), 5.18 (m, 1H), 4.87 (m, 1H), 4.37(m, 1H), 4.19 (m, 1H), 3.31 (s, 3H), 3.24 (m, 1H), 2.83 (m, 1H), 2.45(dd, 1H), 2.22 (dd, 1H), 2.07-1.00 (m, 1 8H), 1.23 (s, 3H), 1.20 (s,3H), 0.88 (s, 18H), 0.83 (d, 3H), 0.58 (s, 3H), 0.11 (s, 9H), 0.06 (m,12H)

EXAMPLES Example 11(S),3(R)-Dihydroxy-20(R)-(1-methoxy-4-hydroxy-4-methyl-1-pentyl)-9,10-seco-pregna-5(Z),7(E)10(19)-triene;isomer A (Compound 101)

Method: General Procedure 5.

Starting material IV: Compound 401.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.52 (s, 3H), 0.84 (d, 3H), 1.22 (s, 3H), 1.23 (s, 3H),1.15-2.10 (m, 21H), 2.31 (dd, 1H), 2.59 (dd, 1H), 2.84 (m, 1H), 3.17 (m,1H), 3.34 (s, 3H), 4.22 (m, 1H), 4.42 (m, 1H), 5.00 (m, 1H), 5.33 (m,1H), 6.02 (d, 1H), 6.37 (d, 1H).

Example 21(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-4-hydroxy-4-methyl-1-pentyl)-9,10-seco-pregna-5(Z),7(E),-10(19)-triene:isomer A (Compound 102)

Method: General Procedure 5.

Starting material IV: Compound 402.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.51 (s, 3H), 0.85 (d, 3H), 1.19 (t, 3H)i 1.22 (s, 3H), 1.23(s, 3H), 1.10-2.10 (m, 21H), 2.31 (dd, 1H), 2.60 (dd, 1H), 2.85 (dd,1H), 3.19 (m, 1H), 3.49 (m, 2H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m,1H), 5.33 (m, 1H), 6.02 (d, 1H), 6.39 (d, 1H).

Example 3 1(S),3(R)-Dihydroxy-20(R)-(4-ethyl-4-hydroxy-1-methoxy-1-hexyl)-9,10-seco-pregna-5(Z),7(E),-10(19)-triene:isomer A (Compound 103)

Method: General Procedure 5.

Starting material IV: Compound 403.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.52 (s, 3H), 0.86 (m, 9H), 1.20-2.10 (m, 25H), 2.32 (dd, 1H),2.60 (dd, 1H), 2.85 (m, 1H), 3.16 (m, 1H), 3.34 (s, 3H), 4.23 (m, 1H),4.43 (m, 1H), 5.00 (m, 1H), 5.33 (m, 1H), 6.02 (d, 1H), 6.38 (d, 1H).

Example 41(S),3(R)-Dihydroxy-20(R)-(5-hydroxy-1-methoxy-5-methyl-1-hexyl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene:isomer A (Compound 104)

Method: General Procedure 5.

Starting material IV: Compound 404.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.52 (s, 3H), 0.83 (d, 3H), 1.22 (s, 6H), 1.20-2.10 (m, 23H),2.32 (dd, 1H), 2.60 (dd, 1H), 2.85 (m, 1H), 3.20 (m, 1H), 3.34 (s, 3H),4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m, 1H), 5.33 (m, 1H), 6.02 (d, 1H),6.38 (d, 1H).

Example 51(S),3(R)-Dihydroxy-20(R)-(5-ethyl-5-hydroxy-1-methoxy-1-heptyl)-9,10-seco-pregna-5(Z),7(E),-10(19)-triene; isomer A (Compound 105)

Method: General Procedure 5.

Starting material IV: Compound 405.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ0.52 (s, 3H), 0.82 (d, 3H), 0.86 (t, 6H), 1.46 (q, 4H),1.20-2.15 (m, 23H), 2.31 (dd, 1H), 2.60 (dd, 1H), 2.84 (m, 1H), 3.20 (m,1H), 3.33 (s, 3H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m, 1H), 5.33 (m,1H), 6.02 (d, 1H), 6.38 (d, 1H).

Example 6 1(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-5-ethyl-5-hydroxy-1-heptyl)-9,10-seco-pregna-5(Z),7(E),-10(19)-triene: isomer A (Compound 106)

Method: General Procedure 6.

Starting material IV: Compound 406.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.51 (s, 3H), 0.83 (d, 3H), 0.86 (t, 6H), 1.18 (t, 3H), 1.46(q, 4H), 1.15-2.15 (m, 23H), 2.31 (dd, 1H), 2.60 (dd, 1H), 2.85 (m, 1H),3.23 (m, 1H), 3.48 (m, 2H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m, 1H),5.33 (m, 1H), 6.02 (d, 1H), 6.38 (d, 1H).

Example 7 1(S),3(R)-Dihydroxy-20(R)-(6-ethyl-6-hydroxy-1-methoxy-1-octyl)-9.10-seco-pregna-5(Z),7(E),-10(19)-triene: isomer A (Compound 107)

Method: General Procedure 6.

Starting material IV: Compound 407.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.52 (s, 3H), 0.82 (d, 3H), 0.86 (t, 6H), 1.46 (q, 4H),1.15-2.10 (m, 25H), 2.31 (dd, 1H), 2.60 (m, 1H), 2.84 (m, 1H), 3.21 (m,1H), 3.33 (s, 3H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m, 1H), 5.33 (m,1H), 6.02 (d, 1H), 6.38 (d, 1H).

Example 8 1(S),3 (R)-Dihydroxy-20(R)-(1-ethoxy-6-ethyl-6-hydroxy-1-octyl)-9,10-seco-pregna-5(Z),7(E),-10(19)-triene: isomer A (Compound 108)

Method: General Procedure 6.

Starting material IV: Compound 408.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=0.51 (5, 3H), 0.82 (d, 3H), 0.85 (t, 6H), 1.18 (t, 3H), 1.46(q, 4H), 1.10-2.10, (m, 25H), 2.31 (dd, 1H), 2.60 (dd, 1H), 2.84 (m,1H), 3.22 (m, 1H), 3.47 (q, 2H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m,1H), 5.33 (m, 1H), 6.02 (d, 1H), 6.38 (d, 1H).

Example 9(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-5-hydroxy-5-methyl-1-hexyl)-9, 10-seco-pregna-5(Z),7 (E),-10(19)-triene: isomer A (Compound 110)

Method: General Procedure 5.

Starting material IV: Compound 410.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹³C NMR: 147.7, 143.0, 133.2, 124.8, 117.0, 111.7, 83.1, 71.0, 70.7,66.7, 64.5, 55.7, 50.6, 46.4, 45.2, 44.1, 42.9, 39.2, 34.9, 31.4, 29.4,29.2, 29.1, 24.9, 23.5, 22.3, 21.3, 15.8, 13.2, 12.9

Example 10 1(S),3(R)-Dihydroxy-20(R)-(6-hydroxy-1-methoxy-6-methyl-1-heptyl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene:isomer A (Compound 111)

Method: General Procedure 5.

Starting material IV: Compound 411.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹³C NMR: 147.7, 143.1, 133.0, 124.9, 117.0, 111.8, 83.8, 71.0, 70.8,66.8, 56.7, 55.8, 51.2, 46.1, 45.3, 44.0, 42.9, 39.6, 35.4, 30.3, 29.3,29.2, 29.1, 27.0, 25.8, 24.7, 23.5, 22.2, 12.8, 12.6

Example 11 (S),3 (R)-Dihydroxy-20(R)-(1-ethoxy-6-hydroxy-6-methyl-1-heptyl)-9. 10-seco-pregna-5(Z),7(E),10(19)-triene: isomer A (Compound 112)

Method: General Procedure 5.

Starting material IV: Compound 412.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=6.38 (d, 1H), 6.01 (d, 1H), 5.33 (m, 1H), 5.00 (m, 1H), 4.43(m, 1H), 4.23 (m, 1H), 3.48 (q, 2H), 3.22 (m, 1H), 2.84 (dd, 1H), 2.60(dd, 1H), 2.32 (dd, 1H), 2.08-1.09 (m, 25H), 1.21 (s, 6H), 1.18 (t, 3H),0.82 (d, 3H), 0.51 (s, 3H)

Example 121(S),3(R)-Dihydroxy-20(R)-(4-hydroxy-4--methyl-1-n-propoxy-1-pentyl)-9.10-seco-pregna-5(Z),7(E),10(19)-triene: isomer A (Compound 114)

Method: General Procedure 5.

Starting material IV: Compound 414.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=6.39 (d, 1H), 6.02 (d, 1H), 5.33 (m, 1H), 5.00 (m, 1H), 4.43(m, 1H), 4.23 (m, 1H), 3.38 (m, 2H), 3.19 (m, 1H), 2.84 (dd, 1H), 2.60(dd, 1H), 2.32 (dd, 1H), 2.10-1.13 (m, 23H), 1.22 (s, 6H), 0.92 (t, 3H),0.85 (d, 3H), 0.51 (s, 3H)

Example 13 1(S),3 (R)-Dihydroxy-20(R)-(1-benzyloxy-4-hydroxy-4-methyl-1-pentyl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene:isomer A (Compound 115)

Method: General Procedure 5.

Starting material IV: Compound 415

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=7.41-7.23 (m, 5H), 6.38 (d, 1H), 6.02 (d, 1H), 5.33 (m, 1H),5.00 (m, 1H), 4.53 (m, 2H), 4.43 (m, 1H), 4.23 (m, 1H), 3.42 (m, 1H),2.83 (dd, 1H), 2.60 (dd, 1H), 2.31 (dd, 1H), 2.11-1.13 (m, 21H), 1.22(s, 6H), 0.90 (d, 3H), 0.53 (s, 3H)

Example 141(S),3(R)-Dihydroxy-20(R)-(4-hydroxy-4--methyl-1-phenoxythiocarbonyloxy-1-pentyl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene;isomer A (Compound 116)

Method: General Procedure 6.

Starting material IV: Compound 416.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=7.42 (t, 2H), 7.29 (t, 1H), 7.08 (d, 2H), 6.39 (d, 1H), 6.03(d, 1H), 5.34 (t, 1H), 5.24 (m, 1H), 5.01 (m, 1H), 4.44 (m, 1H), 4.24(m, 1H), 2.85 (dd, 1H), 2.61 (dd, 1H), 2.32 (dd, 1H), 2.15-1.20 (m,21H), 1.26 (s, 6H), 0.97 (d, 3H), 0.54 (s, 3H)

Example 151(S),3(R)-Dihydroxy-20(R)-(1-methoxy-4-hydroxy-4-methyl-1-pentyl)-9.10-seco-pregna-5(Z),7(E),10(19)-triene: isomer B (Compound 121)

Method: General Procedure 5.

Starting material IV: Compound 421.

Chromatography eluant: 50% to 0% pet.ether in ethylacetate.

¹H NMR: δ=6.38 (d, ₁H), 6.03 (d, 1H), 5.34 (m, 1H), 5.00 (m, 1H), 4.44(m, 1H), 4.24 (m, 1H), 3.32 (s, 3H), 3.30 (m, 1H), 2.84 (m, 1H), 2.61(dd, 1H), 2.32 (dd, 1H), 2.16 (s, 1H), 2.09-1.11 (m, 20H), 1.22 (s, 3H),1.21 (s, 3H), 0.82 (d, 3H), 0.60 (s, 3H)

Example 16 Capsules containing Compound 101

Compound 101 was dissolved in arachis oil to a final concentration of 1μg of Compound 101/ml oil. 10 Parts by weight of gelatine, 5 parts byweight glycerine, 0.08 parts by weight potassium sorbate, and 14 partsby weight distilled water were mixed together with heating and formedinto soft gelatine capsules. These were then filled each with 100 μl ofCompound 101 in oil solution, such that each capsule contained 0.1 pg ofCompound 101.

Example 17 Dermatological Cream Containing Compound 102

In 1 g almond oil was dissolved 0.05 mg of Compound 102. To thissolution was added 40 g of mineral oil and 20 g of self-emulsifyingbeeswax. The mixture was heated to liquify. After the addition of 40 mlhot water, the mixture was mixed well. The resulting cream containsapproximately 0.5 μg of Compound 102 per gram of cream.

The following scientific articles and references have been citedthroughout this application and the entire contents of each article orreference is hereby incorporated by reference.

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Hansen, K. Biochem. Pharmacol. 1991, 42, 1569-1575.

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What is claimed is:
 1. A compound of the formula

in which formula X is hydrogen or hydroxy; R¹ and R², which may be thesame or different, stand for hydrogen or a C₁-C₃ alkyl radical; or R¹and R², taken together with the carbon atom bearing the group X, canform a C₃-C₆ carbocyclic ring; R³ stands for a C₁-C₃ alkyl radical, anaryl or an aralkyl radical, or for YR⁴, in which Y stands for theradicals —CO—S—, —CS—O—or —CS—S—, and R⁴ stands for a C₁-C₃ alkylradical or an aryl or an aralkyl radical; Q is (CH₂)_(n), n being 1-4;R¹, R² and Q independently may optionally be substituted with one ormore fluorine atoms.
 2. A compound of formula I according to claim 1 inwhich Q is (CH₂)n, n being 2 or 3, in which X is hydroxy, R¹ and R² aremethyl or ethyl, and R³ is methyl or ethyl.
 3. A diastereoisomer of acompound according to any one of claims 1 or 2, in pure form; or amixture of said diastereoisomers.
 4. A diastereoisomer of a compoundaccording to claim 3 having the “R”-configuration at C-22.
 5. A compoundaccording to claim 1 which is 1(S),3(R)-Dihydroxy-20(R)-(1-methoxy-4-hydroxy-4-methyl-1-pentyl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene;isomer A, or1(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-4-hydroxy-4-methyl-1-pentyl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene;isomer A.
 6. A method for producing a compound of formula I of claim 1in which a)1(S),3(R)-bis-(tert)-butyldimethylsilyloxy)-20(R)-formyl-9,10-seco-pregna-5(E),7(E),10(19)-trieneis reacted with an organometallic reagent R-Met-Hal or R-Met, in whichMet is a metal, HaI is Cl, Br or I, and R is —Q—CR¹R²—X¹  in which X isH, OH or OR⁵, R⁵ being an alcohol protective group, and R¹, R² and Qhave the above meanings, to form a mixture of two C-22-epimers IIA andIIB

 which epimers are separated; b) a compound of formula IIA or IIB ofstep a), is alkylated to the corresponding compound IIIA or IIIB, inwhich R and R³ have the above meanings or optionally acylated to thecorresponding compound IIIA or IIIB, where R³=YR⁴, Y and R⁴ having themeanings defined in claim 1

c) a compound of formula IIIA or IIIB of step b) is isomerized to thecorresponding compound IVA or IVB by means of UV-light in the presenceof a triplet sensitizer,

 in which R and R³ have the above meanings; d) a compound of formula IVAor IVB is deprotected to the corresponding compound of the generalformula
 1. 7. A method according to claim 6 in which steps b) and c) areperformed in the reverse order.
 8. A method according to claim 6 inwhich steps c) and d) are performed in the reverse order.
 9. Apharmaceutical composition containing an effective amount of one or moreof the compounds of claim 1, together with pharmaceutically acceptablecarriers.
 10. A pharmaceutical composition according to claim 9 indosage unit form containing from 0.1 ppm to 0.1% by weight of the dosageunit of a compound of formula
 1. 11. A method for the treatment ofabnormal skin cell proliferation or for obtaining an immunosuppressiveeffect, said method comprising administering to a patient in need ofsuch treatment an effective amount of a compound according to claim 1.12. A method of treatment for inhibiting undesirable skin cellproliferation which comprises administering to a patient in need of suchtreatment, an effective amount of a compound according to claim 1.